The Swr1 chromatin-remodeling complex prevents genome instability induced by replication fork progression defects.

Genome instability is associated with tumorigenesis. Here, we identify a role for the histone Htz1, which is deposited by the Swr1 chromatin-remodeling complex (SWR-C), in preventing genome instability in the absence of the replication fork/replication checkpoint proteins Mrc1, Csm3, or Tof1. When combined with deletion of SWR1 or HTZ1, deletion of MRC1, CSM3, or TOF1 or a replication-defective mrc1 mutation causes synergistic increases in gross chromosomal rearrangement (GCR) rates, accumulation of a broad spectrum of GCRs, and hypersensitivity to replication stress. The double mutants have severe replication defects and accumulate aberrant replication intermediates. None of the individual mutations cause large increases in GCR rates; however, defects in MRC1, CSM3 or TOF1 cause activation of the DNA damage checkpoint and replication defects. We propose a model in which Htz1 deposition and retention in chromatin prevents transiently stalled replication forks that occur in mrc1, tof1, or csm3 mutants from being converted to DNA double-strand breaks that trigger genome instability

Proficient replication of the yeast genome by a viral DNA polymerase

DNA replication in eukaryotic cells requires minimally three B-family DNA polymerases: Pol α, Pol δ, and Pol ϵ. Pol δ replicates and matures Okazaki fragments on the lagging strand of the replication fork. Saccharomyces cerevisiae Pol δ is a three-subunit enzyme (Pol3-Pol31-Pol32). A small C-terminal domain of the catalytic subunit Pol3 carries both iron-sulfur cluster and zinc-binding motifs, which mediate interactions with Pol31, and processive replication with the replication clamp proliferating cell nuclear antigen (PCNA), respectively. We show that the entire N-terminal domain of Pol3, containing polymerase and proofreading activities, could be effectively replaced by those from bacteriophage RB69, and could carry out chromosomal DNA replication in yeast with remarkable high fidelity, provided that adaptive mutations in the replication clamp PCNA were introduced. This result is consistent with the model that all essential interactions for DNA replication in yeast are mediated through the small C-terminal domain of Pol3. The chimeric polymerase carries out processive replication with PCNA in vitro; however, in yeast, it requires an increased involvement of the mutagenic translesion DNA polymerase ζ during DNA replication

Upstream-binding factor is sequestered into herpes simplex virus type 1 replication compartments

Previous reports have shown that adenovirus recruits nucleolar protein upstream-binding factor (UBF) into adenovirus DNA replication centres. Here, we report that despite having a different mode of viral DNA replication, herpes simplex virus type 1 (HSV-1) also recruits UBF into viral DNA replication centres. Moreover, as with adenovirus, enhanced green fluorescent protein-tagged fusion proteins of UBF inhibit viral DNA replication. We propose that UBF is recruited to the replication compartments to aid replication of HSV-1 DNA. In addition, this is a further example of the role of nucleolar components in viral life cycle

Reversal of terminal differentiation and control of DNA replication

DNA replication in mammalian cells occurs in discrete nuclear foci. Here we show that terminally differentiated myotubes can be induced to reenter S phase and show the same pattern of replication foci as cycling cells. We used this cellular system to analyze the interaction of cell cycle proteins with these foci in vivo. Cyclin A and cdk2, but not cyclin B1 and cdc2, were specifically localized at nuclear replication foci, just like the replication protein proliferating cell nuclear antigen. A potential target of cyclin A and cdk2 is the 34 kd subunit of replication protein A (RPA34). In contrast with the 70 kd subunit, which localizes to the foci, RPA34 was not detected at these replication sites, which may reflect a transient interaction. The specific localization of cyclin A and cdk2 at nuclear replication foci provides a direct link between cell cycle regulation and DNA replication

Checkpoints are blind to replication restart and recombination intermediates that result in gross chromosomal rearrangements

Replication fork inactivation can be overcome by homologous recombination, but this can cause gross chromosomal rearrangements that subsequently missegregate at mitosis, driving further chromosome instability. It is unclear when the chromosome rearrangements are generated and whether individual replication problems or the resulting recombination intermediates delay the cell cycle. Here we have investigated checkpoint activation during HR-dependent replication restart using a site-specific replication fork-arrest system. Analysis during a single cell cycle shows that HR-dependent replication intermediates arise in S phase, shortly after replication arrest, and are resolved into acentric and dicentric chromosomes in G2. Despite this, cells progress into mitosis without delay. Neither the DNA damage nor the intra-S phase checkpoints are activated in the first cell cycle, demonstrating that these checkpoints are blind to replication and recombination intermediates as well as to rearranged chromosomes. The dicentrics form anaphase bridges that subsequently break, inducing checkpoint activation in the second cell cycle

Genetic and physical mapping of DNA replication origins in Haloferax volcanii

The halophilic archaeon Haloferax volcanii has a multireplicon genome, consisting of a main chromosome, three secondary chromosomes, and a plasmid. Genes for the initiator protein Cdc6/Orc1, which are commonly located adjacent to archaeal origins of DNA replication, are found on all replicons except plasmid pHV2. However, prediction of DNA replication origins in H. volcanii is complicated by the fact that this species has no less than 14 cdc6/orc1 genes. We have used a combination of genetic, biochemical, and bioinformatic approaches to map DNA replication origins in H. volcanii. Five autonomously replicating sequences were found adjacent to cdc6/orc1 genes and replication initiation point mapping was used to confirm that these sequences function as bidirectional DNA replication origins in vivo. Pulsed field gel analyses revealed that cdc6/orc1-associated replication origins are distributed not only on the main chromosome (2.9 Mb) but also on pHV1 (86 kb), pHV3 (442 kb), and pHV4 (690 kb) replicons. Gene inactivation studies indicate that linkage of the initiator gene to the origin is not required for replication initiation, and genetic tests with autonomously replicating plasmids suggest that the origin located on pHV1 and pHV4 may be dominant to the principal chromosomal origin. The replication origins we have identified appear to show a functional hierarchy or differential usage, which might reflect the different replication requirements of their respective chromosomes. We propose that duplication of H. volcanii replication origins was a prerequisite for the multireplicon structure of this genome, and that this might provide a means for chromosome-specific replication control under certain growth conditions. Our observations also suggest that H. volcanii is an ideal organism for studying how replication of four replicons is regulated in the context of the archaeal cell cycle. © 2007 Norais et al

DNAReplication: a database of information and resources for the eukaryotic DNA replication community

DNAReplication (at http://www.dnareplication.net) has been set up as a freely available single resource to facilitate access to information on eukaryotic DNA replication. This database summarizes organism-sorted data on replication proteins in the categories of nomenclature, biochemical properties, motifs, interactions, modifications, structure, cell localization and expression, and general comments. Replication concepts are defined and a general model of the steps in DNA replication is presented. Links to relevant websites and homepages of replication labs are provided. The site also has an interactive section where links to recent replication papers are posted and readers are provided with the facility to post comments about each paper. The interactive and links pages are modified weekly and the whole site is updated annually